Production of 6-aminopenicillanic acid



PRODUCTION OF 6-AMHVOPENKCILLANIC ACID George Newbolt Rolinson,Parlrgate House, Newdigate, England; Merfyn Richards, Flat 1, BrochharnPark, Betchworth, England; and Frank Ralph Batclrelor, The

Flat, Gatton Manor, Ockley, England No Drawing. Filed Jan. 25, 1960,Ser. No. 4,236 4 Claims. (Cl. 195-36) This invention relates to chemicalprocesses for the production of substances having antibiotic activityand which are useful in the preparation of novel penicillins and, moreparticularly, relates to an improvement in processes for the productionof 6-aminopenicillanic acid by the enzymatic degradation of certainpenicillins.

Various processes for the production and recovery of 6-aminopenicilanicacid, which has the following structural formula are described inBelgian Patent No. 569,728 and in the scientific literature,particularly in a publication by Batchelor et al. in Nature 183, pages257-258 (1959). In such processes a penicillin-producing mould is grownin a nutrient medium and the 6-aminopenicillanic acid or a salt thereofis isolated from the fermentation liquor obtained.

It has recently become known that 6-aminopenicillanic acid can be usedto produce infinite numbers of novel penicillins by its reaction withacylating agents such as acid chlorides and acid anhydrides. Many suchnovel penicillins have already exhibited potent antibacterial activityand some have given promise of surprising attributes such as enhancedactivity against resistant organisms and resistance to destruction byacid and by penicillinase. Known methods of producing6-aminopenicillanic acid are very difiicult to execute, particulary in acommercially feasible manner in which the economical production of l rgequantities of the product is required. The synthetic production of6-aminopenicillanic acid on a practical commercial scale has yet to beachieved.

Prior workers in the art have suggested that a pencillin product in afermentation medium is not a stable product but rather is in a state ofdynamic equilibrium with other penicillin products present in thefermentation medium (Wideburg and Peterson Abstract of Papers of Am.Chem. Soc., 132nd meeting, September 8l3, 1957). Other workers(Sakaguchi et al. cited below) suggested that penicillin G could beenzymatically hydrolyzed to produce penicin, a compound for which thestructure of 6-aminopenicillanic acid was suggested. 6-aminopenicillanicacid, however, was either not obtained by any of these workers or wasobtained in such impure form and minute quantities that it could not becharacterized.

It is therefore an object of the present invention to provide animproved process for producing 6-aminopenicillanic acid. It is aparticular object of the present invention to provide a process forproducing 6-aminopenicillanic acid by the enzymatic degradation ofcertain penicilins.

We have now found that certain penicillins can be converted to6-aminopenicillanic acid by means of an enzyme or mixture of enzymesreferred to herein as pencillin amidase. Accordingly, there is providedby the present invention a process for the preparation of6-aminopenicillanic acid and salts thereof which comprises treating apenicillin with a penicillin amidase, said penicillin being selectedfrom the group consisting of the penicillins having the formula whereinX is a member selected from the group consisting of sulphur and oxygen,R is a member selected from the group consisting of alkyl groups havingfrom 2 to 6 carbon atoms inclusive; alkenyl groups having from 3 to 6carbon atoms inclusive; phenyl; and monosubstituted phenyl groups inwhich the substituent is a member selected from the group consisting ofchloro, bromo, iodo, fiuoro, nitro, alkyl groups having from 1 to 6carbon atoms inclusive, alkenyl groups having from 3 to 6 carbon atomsinclusive, and alkoxy in which the alkyl group has from 1 to 6 carbonatoms inclusive.

The term alkyl as used herein means both branched and straight chainsaturated aliphatic hydrocarbon groups such as, for example, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, n-amyl, isoarnyl, etc., and boththe branched and straight chain saturated aliphatic radicals may have ahalogen substituent such as Z-chlorocthyl etc. The term alkenyl as usedherein means both branched and straight chain unsaturated aliphtichydrocarbon radicals having from 3 to 6 carbon atoms inclusive such as,for example, allyl, 2-rnethyl allyl, butene-2-yl, and themonosubstituted branched and straight chain unsaturated aliphaticradicals wherein the monosubstituent is chloro, fluoro, iodo or bromosuch as 2-bromoallyl, S-chloroallyl, etc.

The penicillins of Formula II above which are used as substrates in theprocess of the present invention include those penicillins produced by afermentation process using a precursor as described in US. Patents Nos.2,562,410, and 2,479,295, and 2,479,296, and 2,854,450 relating to thepreparation of many biosynthetic penicillins. Such penicillins include,for example, phenoxymethyl penicillin (penicillin V) and monosubstitutedphenoxymethyl penicillins such as p-methoxyphenoxymethyl penicillin,pnitrophenoxymethyl penicillin, hydroxypnenoxymethyl penicillin,p-chlorophenoxymethyl penicillin, pand mtoloxyphenoxymethyl penicillin,etc.; alkoxyrnethyl penicillins such as, for example, ethoxymethylpenicillin, nbutoxymethyl penicillin, isoamyloxymethyl penicillin,

hexyloxylmethyl penicillin; alkenyloxymethyl penicillins uch asallyloxymethyl penicillin, Z-methylallyloxymethyl penicillin, etc.; andthe sulfur-containing penicillins such as p-tolylthiomethyl penicillin,B-naphthylthiomethyl penicillin, ethylthiomethyl penicillin,n-butylthiomethyl penicillin, allylthiomethyl penicillin, etc. Each ofthe penicillins of Formula II can housed in the form of their salts witha cation which will not interfere with the enzymatic hydrolysis; themost common of such salts are the alkali metal and alkaline earth metalsalts such as the sodium, calcium and potassium salts.

One penicillin amidase, i.e., one enzyme preparation which may beemployed in the process of the present invention, is -a known enzyme ormixture of enzymes, the preparation of which in the form of a solid andactive aqueous solution is described by Kinichiro Sakaguchi and SawaoMurao in I. Agr. Chem. Soc. Japan 23, page 411, (1950) [see also CA. 45,1197, (1951)] and in greater detail by Sawao Murao in I. Agr. Chem. Soc.Japan, 29,

pages 400-407 (1955), which is available in the English ration ofpenicillin amidase, both Sakaguchi and Murao used Penicilliumchrysogenum Q176, a culture which has been widely used in the penicillinindustry. [See the paper entitled Separation and Identification ofPenicillin Species in Commercial Penicillin (Q-l76 Culture) presented byC. J. Salivar et al. at the Conference on Antibiotic Research,Washington, D.C., January 31 and February 1, 1947]. A preferred culture,a strain of Streptomyces lavendulae, employed in the preparation ofpenicillin amidase is on deposit in the American Type Culture Collectionat Washington, D.C., wherein it is identified as ATIC No. 13664. Anotherdeposited culture, ATCC No. 13665, generally produces less pigment thanATCC No. 13664 and can also be employed to produce penicillin amidase.

We have also found that the fungi Botryris cinerea and Penicilliumchrysogenum as well as certain other fungi of the genera Alternaria,Aspergillus, Epicoccum, Fusarium, Mucor, Penicillium, Phoma andTrichoderma, the yeasts Crypt coccus albidus and a species ofTrichosporon, and several Actinomycetes of the genus Streptomycesproduce penicillin amidase. Penicillin amidase can be produced frompenicillin amidase-producing micro-organisms of the genus Streptomycesby the following general procedure which is also illustrated by thespecific procedure set forth in greater detail below.

A suitable enzyme preparation for the process of the present inventioncan be obtained by the following gen eral procedure. First generationslopes on nutrient agar are first prepared from a soil stock of sporesof a penicillin amidase-producing microorganism of the genusStreptomyces. After inoculation permitting suitable growth andsporulation to take place, these first generation slopes form theinoculum for the second generation slopes of the same culture medium.Adequately sporulated second generation slopes are washed with Sterilewater and the resulting suspension of spores aseptically transferred tothe seed vessel containing nutrient broth. When this fermentation hasrun for from 24 to 48 hours the brew is transferred to a larger batch ofnutrient broth. After agitation and aeration for a period of 2 to 4 daysthe mycelium is removed to leave a clear fermentation liquor containingpenicillin amidase. The enzyme can be precipitated, if desired, with 90%saturated ammonium sulfate solution.

In another procedure which is described in detail in Example 4 below,penicillin amidase is prepared by the fermentation of a culture ofBotrytis cinerea. In general in this procedure a suitable enzymepreparation may be obtained by using a culture of the organism toinoculate a seed vessel containing a suitable seed culture medium. Apenicillin is added aseptically to the seed medium at inoculation. Theseed stage is allowed to grow for 24-48 hours after which the whole brewbecomes the inoculum for the final fermentation. A portion of the seedis inoculated into a larger batch of growth medium. A penicillin is alsoadded aseptically to the growth medium at inoculation. These additionsof penicillin are made to promote formation of the penicillin amidaseenzyme. After allowing the final fermentation to run for a period oftime a quantity of the penicillin is added for conversion to6-aminopenicillanic acid. The pH at which the reaction is carried outdepends on the particular organism used. At the end of the reaction timethe liquid is separated from the organism and any solid residues of theculture medium and the 6-aminopenicillanic acid may be recovered fromthe aqueous solution.

In experiments relating to the stability of penicillin amidase in aclarified fermentation broth at various pHs, it is found that after fivehours storage at room temperature at pH 3, 4, 7, 9, 10 and 11 theenzymatic activity remaining in the broth is 2, 24, 85, 87, 100 and 96percent, respectively, of the original enzymatic activity. In analogousexperiments also relating to the stability of penicillin amidase inclarified fermentation broth at various temperatures, it is found thatafter 24 hours storage at 5, 18, 35, 45, and 60 C. the enzymatic 4activity remaining in the broth is 100, 90, 76, 67, 51, and 14 percent,respectively, of the original enzymatic activity.

The reaction between the penicillin and the enzyme preparation can takeplace by adding the penicillin to the brew or, preferably, by adding asolution of the penicillin to the dialyzed enzyme. The dialyzed enzymeis prepared by treating the clarified liquor containing the enzyme withammonium sulfate, removing the precipitate which is formed andredissolving it in water and subsequently dialyzing it against runningtap water.

The optimum pH for the enzymatic hydrolysis of penicillins in theproduction of 6-aminopenicillanic acid is about 9.0. Higher and lowerpHs are operable, however, and we have found that penicillin amidase isabout 75% effective at pH 7.5 and 11.0 (based on effectiveness inenzymatic hydrolysis at pH 9.0). As the enzyme reaction mixture becomesmore alkaline than pH ll or more acid than pH 7.5, the productivity ofthe process decreases rapidly. Below pH 6.0 the enzyme is almostcompletely inactive. We prefer to carry out the enzymatic hydrolysis ofthe various penicillin substrates at a temperature of from about 26 to50 C. though somewhat higher and lower temperature, e.g., of the rangeof about 25 to 60 C. can be employed. In most instances the hydrolysisis completed in from 2 to 5 hours though longer periods, as in Example 1below, can be employed. The course of the hydrolysis reaction can befollowed by various assay procedures in order to determine thecompletion of the hydrolysis reaction.

After the completion of the enzymatic hydrolysis, the unreactedpenicillins and the aliphatic carboxylic acid which is split off duringthe reaction are removed by extraction with an organic solvent, e.g.,butyl acct-ate, at an acid pH. Further purification may be affected byneutralizing the aqueous solution and precipitating impurities by addinga solvent such as acetone, methanol or ethanol and then centrifuging toremove the precipitated impurities. The clarified solution is adjustedto pH 6.5 to 7 and passed over an ion exchange resin to adsorb theproduct with or without prior concentration. We prefer to use a columnof a highly basic anion exchange resin, in the chloride form, of across-linked polystyrene type with quaternary ammonium functional groupssuch as Dowex I and DeAcedite FF, both of which are commerciallyavailable. A cross-linked polystyrene cationic exchange resin withsulphonic acid functional groups such as Amberlite IR 120 can also beused. In the absorption step we prefer to use about 4 kg. of resin per15 liters of solution containing from 40 to 60 gm. of6-aminopenicillanic acid. Elution of the product from the resin isaccomplished by the addition of hydrochloric acid, e.g., 0.05 N HCl.Procedures for the recovery of 6-aminopenicillanic acid from aqueouessolutions are also illustrated by Belgian Patent No. 569,728.

PREPARATION OF PENICILLIN AMID-ASE A culture of Streptomyces A.T.C.C.No. 13664 is grown in shaken flasks, 10-liter and -liter fermentersusing a medium consisting of a broth at a concentration of 20 g. perliter and pH 7.3 to 7.4. The broth is of the following composition:

Percent Liquid glucose 3.0 Soya bean meal 2.5 Yeast extract (Difco) 0.5Sodium chloride 0.5

Calcium carbonate 0.2

final fermentation. This final fermentation is allowed to run for 96hours at 26 to 28 C. with stirring and aeration, the mycelium is thenremoved by filtration (or by centrifugation, if desired) and the clearliquor which contained the enzyme is treated with ammonium sulphate togive 90% saturation (650 g./l.). The precipitate is then removed bycentrifuging and redissolved in a small volume of water and dialyzedagainst running tap water for two days. This enzyme preparation is thenfreeze dried. Alternatively, the aqueous enzyme preparation can bestored at 20 C.

A fermentation media composed of:

Grams per ml.

Soya bean meal 40 Cornmeal 40 (NH SO 1 CaCO is also useful in theproduction of penicillin amidase and is used in place of that set forthabove in this procedure; this medium is preferably adjusted to pH 7.2 to7.4 before autoclaving.

The following specific examples will serve to illustrate the process ofthe present invention.

EXAMPLE 1 Production of crystalline o-aminopenicillanic acid To 700 ml.of the dialyzed penicillin amidase prepared as described in theprocedure above is added g. of phenoxymethylpenicillin (penicillin V)dissolved in 50 ml. of Water to give a final concentration of mg./ml.The mixture is incubated at 34 C., the pH being maintained at 8.7 by theperiodic addition of aqueous N caustic soda solution. After 9 hours thereaction mixture is adjusted to pH 2 with phosphoric acid and theprecipitate which is formed is removed by centrifugation. The liquid isthen extracted with a half volume of butyl acetate to remove theresidual phenoxymethyl penicillin, together with the phenoxyacetic acidside chain which is split off during the formation of the6-aminopenicillanic acid. After neutralization with dilute aqueouscaustic soda solution, the resulting liquid is percolated through acolumn of a highly basic anion exchange resin (in the chloride form) ofthe cross-linked polystyrene type with quaternary ammonium functionalgroups (e.g., Dowex I, a commercially available resin of the foregoingdescription) and eluted, concentrated and crystallized as described inBelgian Patent 569,728. The final yield of solid 6-aminopenicillanicacid is 1.0 gram.

Crystalline 6-aminopenicillanic acid is obtained directly from theamidase enzymation liquor without the use of ion exchange resin, byacidifying 200 ml. of amidase reaction mixture containing 820 mg. of6-aminopenicillanic acid to pH 2.0. The precipitate which forms isremoved by centrifugation and the liquid is then extracted with 100 ml.of n-butyl acetate. The aqueous phase is collected and adjusted to pH6.5 with sodium hydroxide and evaporated to 40 ml. Acidifying with 5 NHCl and cooling to 5 C. gives a crop of crystals of 6-aminopenicillanicacid which can be collected in the usual way. These crystals were washedwith a little cold water followed by dry acetone and dried in vacuo togive a yield of 390 mg.

The foregoing process is repeated using butoxymethyl penicillin andp-chlorophenoxymethyl penicillin in place of phenoxymethyl penicillin.The rate of hydrolysis in each case is somewhat increased over the rateof hydrolysis of phenoxy methyl penicillin and the yield in each case isequivalent.

EXAMPLE 2 Production of 6-aminopenicillanic acid from penicillins V andG and n-butylthiomethyl penicillin In order to illustrate thecomparative activity of penicillin amidase on penicillin V andn-butylthiomethyl penicillin as compared with penicillin G, thefollowing procedure is adopted.

The penicillins being compared are each dissolved separately in 0.5 Naqueous sodium bicarbonate solution to give solutions of 10 mg./ ml. andthe pH of each solution is adjusted to 8.7. One ml. of each suchsolution is then treated with 1 ml. of the enzyme solution (prepared asdescribed in the procedure above) at pH 8.7 and the mixture is incubatedat 34 C. in a water bath for 30 minutes. At the end of this time asample of each mixture is spotted on chromatography tape (Whatman No. 4)and run for 3 to 4 hours in a butanol/ethanol/Water, 421:5, system toseparate the unconverted penicillin. Tapes are also spotted withconcentrations of standards of 6-aminopenicillanic acid and similarlytreated. After chromatography the tapes are sprayed with phenylacetylchloride and sodium bicarbonate (producing benzylpenicillin) and areplaced on agar plates seeded with spores of Bacillus subtilis andincubated at 37 C. for development of zones of inhibition. The6-aminopenicillanic acid produced from each of the penicillins is thenestimated against the standards in the conventional bioassay manner. Theresults are expressed in the following table as units/ml. ofo-aminopenicillanic acid produced in the mixture of enzyme andsubstrate.

UNITS/ML. OF G-AMINOPENIOILLANIG ACID PRODUCED Penicillin n-Butyl-Penicillin V thiomethyl G Penicillin 101. Brew 380 370 10 Shaken flaskbrew 5 EXAMPLE 3 A fermentation broth containing penicillin amidase isprepared as set forth in the procedure above and the mycelium is removedby centrifugation. One volume of acetone is then added with stirring tothe clarified broth and the resulting precipitate, penicillin amidase,is then collected by centrifugation. The precipitated penicillin amidaseis then dissolved in Water to give a volume equivalent to one-third ofthe original broth and the resulting solution of the enzyme is thenadjusted to pH 8.5 and to 45 C. Phenoxymethyl penicillin is then addedto the solution to a concentration of 10 gm./liter. The solution ismaintained at pH 8.5 and 45 C. until assay shows no further increase inthe 6-aminopenicillanic acid content. (NOTE.In most reactions this takesfrom 2 to 5 hours.) The remaining penicillins and phenoxyacetic acid(which is produced by the enzymatic hydrolysis) in the reaction mixtureare then removed by solvent extraction with butylacetate. After thesolvent extraction the reaction mixture is cooled to l520 C. and the pHis adjusted to 4.5 with phosphoric acid and centrifuged to removeimpurities. The clear solution is then adjusted to a pH with the rangeof 6.5 to 7.0 and the 6-arninopenicillanic acid collected on an ionexchange column containing a highly basic anion exchange resin (in thechloride form) of the cross-linked polystyrene type with quaternaryammonium functional groups (Dowex I) in an amount equal to 4 kg. ofresin per 15 liter of clarified reaction mixture containing 40-60 gm. of6-aminopenicillanic acid is preferred in this recovery step.

EXAMPLE 4 In another method for producing a fermentation liquoicontaining useful quantities of penicillin amidase and using suchpenicillin amidase to produce o-aminopeniaeigsae d cillanic acid, aculture of Botrytis cinerea B.R.L. No. 36, which is on deposit with theAmerican Type Culture Collection and is identified as ATCC No. 13742, isgrown in shaken flasks using a culture medium made from 50 gms. maizemeals and 1 g. ammonium sulfate in one liter of tap water; the medium isadjusted to pH 6.8 before sterilizing. A seed stage is employed in whichthe organism is grown for about thirty hours at 26 C. in a culturemedium consisting of 80 ml. corn steep liquor and 40 gms. glucose madeup to 1 liter with tap water, the medium being adjusted to pH 6.4 beforesterilizing.

Approximately 7% by volume of the seed stage fermentation is inoculatedinto the growth medium. A penicillin of the type used as substrate inthe production of G-aminopenicillanic acid is added aseptically to theseed stage fermentation and to the growth medium at inoculation in atotal amount equal to about from 250 to 2500 units/ml. in the finalgrowth medium in order to promote the formation of penicillin amidase.This final fermentation is allowed to run for from about 120 to 144hours at which time the concentration of penicillin amidase in the brothis at about the maximum and penicillins may be added to the broth forconversion to 6- aminopenicillanic acid.

In order to illustrate the activity of the enzyme using the substratesof the present invention compared with penicillin G, penicillins as setforth in the table below are separately added aseptically in sterilesolution to a Botrytis cinerea fermentation broth of 120-144 hours agewhich is prepared as described above. A convenient final concentrationof penicillin in the broth is 5,000 units/ml.

After a further 24 hours fermentation, samples of each broth areseparated from their mycelial and solid content and measured volumes ofeach sample placed on chromatography tape for comparative bioassay as inExample 2.

The results are expressed below as units/ml. of 6- zyme and substrate.

n-Butylthiomethyl Pen icillins Penicillin G We claim:

1. A process for the preparation of an aqueous solution of6-aminopenicillanic acid which comprises in sequence (1) growing aculture of Srreptomycas lavena'ulae in a suitable liquid nutrient growthmedium for a period of 2 to 6 days whereby fermentation broth isproduced, (2) separating the major portion of the solid materials fromsaid fermentation broth, (3) mixing phenoxymethyl penicillin with saidfermentation broth to form an aqueous reaction mixture and (4)maintaining said aqueous reaction mixture at a temperature from about 25to 60 C. at a pH of from about 6 to 11 for a period of from about 2 toabout 24 hours.

2. A process for the preparation of an aqueous solution of-aminopenicillanic acid which comprises in sequence (1) growing aculture of Bozrytis cinerea in a suitable liquid nutrient growth mediumfor a period of 2 to 6 days whereby a fermentation broth is produced,

(2) mixing phenoxymethyl penicillin with said fermenta- 6 tion broth toform an aqueous reaction mixture and (3) maintaining said aqueousreaction mixture at a temperature from about 25 to 60 C. at a pH of fromabout 6 to 11 for a period of from about 2 to about 24 hours.

3. A process for the preparation of an aqueous solution of6-aminopenicillanic acid which comprises in sequence (1) growing aculture of Streptomyces lavendulae in a suitable liquid nutrient growthmedium for a period of 2 to 6 days whereby fermentation broth isproduced, (2) separating the major portion of the solid materials fromsaid fermentation broth, (3) mixing a penicillin substrate with saidfermentation broth to form an aqueous reaction mixture and (4)maintaining said aqueous reaction mixture at a temperature from about 25to 60 C. at a pH of from about 6 to 11 for a period of from about 2 toabout 24 hours, said penicillin substrate being selected from the groupconsisting of the penicillins having the formula wherein X is a memberselected from the group consisting of sulfur and oxygen, R is a memberselected from the group consisting of alkyl groups having from 2 to 6carbon atoms inclusive; alkenyl groups having from 3 to 6 carbon atomsinclusive; phenyl; and monosubstituted phenyl in which the substituentis a member selected from the group consisting of chloro, bromo, iodo,fluoro, nitro, alkyl groups having from 1 to 6 carbon atoms inclusive,alkenyl groups having from 3 to 6 carbon atoms inclusive, and alkoxy inwhich the alkyl group has from 1 to 6 carbon atoms inclusive.

4. A process for the preparation of an aqueous solution of6-aminopenicillanic acid which comprises (1) growing a culture ofBotrytis cinerea in a suitable liquid nutrient growth medium for aperiod of 2 to 6 days whereby a fermentation broth is produced, (2)mixing a penicillin substrate with said fermentation broth to form anaqueous reaction mixture, (3) maintaining said aqueout reaction mixtureat a temperature from about 25 to 60 C. at a pH of from about 6 to 11for a period of from about 2 to about 24 hours and (4) recovering6-aminopenicillanic acid from said aqueous reaction mixture, saidpenicillin substrate being selected from the group consisting of thepenicillins having the formula wherein X is a member selected from thegroup consisting of sulfur and oxygen, R is a member selected from thegroup consisting of alkyl groups having from 2 to 6 carbon atomsinclusive; alkenyl groups having from 3 to 6 carbon atoms inclusive;phenyl; and monosubstituted phenyl in which the substituent is a memberselected from the group consisting of chloro, bromo, iodo, fluoro,nitro, alkyl groups having from 1 to 6 carbon atoms inclusive, alkenylgroups having from 3 to 6 carbon atoms inclusive, and alkoxy in whichthe alkyl group has from 1 to 6 carbon atoms inclusive.

References Cited in the file of this patent J. Agr. Chem. Soc. Japan,23, page 411 (1950). Nature 183, pages 257-258, January 24, 1959.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent .No, 3 014846 lleicember 26 1961 George Newbolt Rolinson et alo It is herebycertified that error appears in the above numbered patent requiringcorrection and that the said Letters Patent should read as correctedbelow.

, In the heading to the printed specification between line 7 and 8insert the following:

Claims priority application Great Britain Jan, ,.3O 195? column 2 line26 for aliphtic read aliphatic column 5 line 61 for "were" read areSigned and sealed this 1st day of May 1962,

(SEAL) Attest:

ERNEST we SWIDER DAVID D Attesting Officer Commissioner of Patents

1. A PROCESS FOR THE PREPARATION OF AN AQUEOUS SOLUTION OF6-AMINOPENICILLANIC ACID WHICH COMPRISES IN SEQUENCE (1) GROWING ACULTURE OF STREPTOMYCES LAVENDULAE IN A SUITABLE LIQUID NUTRIENT GROWTHMEDIUM FOR A PERIOD OF 2 TO 6 DAYS WHEREBY FERMENTATION BROTH ISPRODUCED, (2) SEPARATING THE MAJOR PORTION OF THE SOLID MATERIALS FROMSAID FERMENTATION BROTH, (3) MIXING PHENOXYMETHYL PENICILLIN WITH SAIDFERMENTATION BROTH TO FORM AN AQUEOUS REACTION MIXTURE AND (4)MAINTAINING SAID AQUEOUS REACTION MIXTURE AT A TEMPERATURE FROM ABOUT 25TO 60*C. AT A PH OF FROM ABOUT 6 TO 11 FOR A PERIOD OF FROM ABOUT 2 TOABOUT 24 HOURS.